Biliary Cholesterol Saturation Research Articles

Glutaredoxin 1 regulates cholesterol metabolism and gallstone formation by influencing protein S-glutathionylation

ObjectiveCholesterol gallstone disease (CGD) is closely related to cholesterol metabolic disorder. Glutaredoxin-1 (Glrx1) and Glrx1-related protein S-glutathionylation are increasingly being observed to drive various physiological and pathological processes, especially in metabolic diseases such as diabetes, obesity and fatty liver. However, Glrx1 has been minimally explored in cholesterol metabolism and gallstone disease. MethodsWe first investigated whether Glrx1 plays a role in gallstone formation in lithogenic diet-fed mice using immunoblotting and quantitative real-time PCR. Then a whole-body Glrx1-deficient (Glrx1−/−) mice and hepatic-specific Glrx1-overexpressing (AAV8-TBG-Glrx1) mice were generated, in which we analyzed the effects of Glrx1 on lipid metabolism upon LGD feeding. Quantitative proteomic analysis and immunoprecipitation (IP) of glutathionylated proteins were performed. ResultsWe found that protein S-glutathionylation was markedly decreased and the deglutathionylating enzyme Glrx1 was greatly increased in the liver of lithogenic diet-fed mice. Glrx1−/− mice were protected from gallstone disease induced by a lithogenic diet because their biliary cholesterol and cholesterol saturation index (CSI) were reduced. Conversely, AAV8-TBG-Glrx1 mice showed greater gallstone progression with increased cholesterol secretion and CSI. Further studies showed that Glrx1-overexpressing greatly altered bile acid levels and/or composition to increase intestinal cholesterol absorption by upregulating Cyp8b1. In addition, liquid chromatography-mass spectrometry and IP analysis revealed that Glrx1 also affected the function of asialoglycoprotein receptor 1 (ASGR1) by mediating its deglutathionylation, thereby altering the expression of LXRα and controlling cholesterol secretion. ConclusionOur findings present novel roles of Glrx1 and Glrx1-regulated protein S-glutathionylation in gallstone formation through the targeting of cholesterol metabolism. Our data advises Glrx1 significantly increased gallstone formation by simultaneously increase bile-acid-dependent cholesterol absorption and ASGR1- LXRα-dependent cholesterol efflux. Our work suggests the potential effects of inhibiting Glrx1 activity to treat cholelithiasis.

Effect of hepatic NPC1L1 on cholesterol gallstone disease and its mechanism

Cholesterol gallstone disease (CGD) is associated with bile cholesterol supersaturation. The Niemann-Pick C1-like 1 (NPC1L1), the inhibitory target of ezetimibe (EZE), is a critical sterol transporter of cholesterol absorption. Intestinal NPC1L1 facilitates the absorption of cholesterol, whereas hepatic NPC1L1 promotes cholesterol uptake by hepatocytes and reduces bile cholesterol supersaturation. The potential of hepatic NPC1L1 to prevent CGD has yet to be established due to its absence in the mice model. In this study, we generated mice expressing hepatic NPC1L1 using adeno-associated virus (AAV) gene delivery. The biliary cholesterol saturations and gallstone formations were explored under chow diet and lithogenic diet (LD) with or without EZE treatment. The long-term (8-week) LD-fed AAV-mNPC1L1 mice exhibited no significant differences in biliary cholesterol saturation and gallstone formation compared to WT mice. EZE effectively prevented CGD in both WT and AAV-mNPC1L1 mice. Mechanistically, prolonged LD feeding induced the degradation of hepatic NPC1L1, whereas short-term (2-week) LD feeding preserved the expression of hepatic NPC1L1. In conclusion, our findings suggest that hepatic NPC1L1 is unable to prevent CGD, whereas EZE functions as an efficient bile cholesterol desaturator during CGD development.

FGF15 promotes hepatic NPC1L1 degradation in lithogenic diet-fed mice

BackgroundCholesterol gallstone disease (CGD) is accompanied by biliary cholesterol supersaturation. Hepatic Niemann-Pick C1-like 1 (NPC1L1), which is present in humans but not in wild-type (WT) mice, promotes hepatocyte cholesterol uptake and decreases biliary cholesterol supersaturation. In contrast, intestinal NPC1L1 promotes intestinal cholesterol absorption, increasing biliary cholesterol supersaturation. Ezetimibe (EZE) can inhibit both hepatic and intestinal NPC1L1. However, whether hepatic NPC1L1 can affect CGD progress remains unknown.MethodsMice expressing hepatic NPC1L1 (NPC1L1hepatic-OE mice) were generated using Adeno-associated viruses (AAV) gene delivery. The protein level and function of hepatic NPC1L1 were examined under chow diet, high fat-cholesterol diet (HFCD), and lithogenic diet (LD) feeding. Gallstone formation rates were examined with or without EZE treatment. Fibroblast growth factor 15 (FGF15) treatment and inhibition of fibroblast growth factor receptor 4 (FGFR4) were applied to verify the mechanism of hepatic NPC1L1 degradation.ResultsThe HFCD-fed NPC1L1hepatic-OE mice retained the biliary cholesterol desaturation function of hepatic NPC1L1, whereas EZE treatment decreased biliary cholesterol saturation and did not cause CGD. The ubiquitination and degradation of hepatic NPC1L1 were discovered in LD-fed NPC1L1hepatic-OE mice. Treatment of FGF15 during HFCD feeding and inhibition of FGFR4 during LD feeding could affect the protein level and function of hepatic NPC1L1.ConclusionsLD induces the ubiquitination and degradation of hepatic NPC1L1 via the FGF15-FGFR4 pathway. EZE may act as an effective preventative agent for CGD.

Antilithogenic and hypocholesterolemic effect of dietary fenugreek seeds (Trigonella foenum-graecum) in experimental mice

Fenugreek (Trigonella foenum-graecum) is being used as food adjuncts to enhance the organoleptic properties of foods. Several health beneficial attributes of fenugreek seeds have been evidenced in earlier studies. Thus objective of the investigation was to evaluate the hypocholesterolemic and antilithogenic property of fenugreek seeds. Cholesterol gallstones were induced in mice by feeding high cholesterol diet (0.5% cholesterol and 0.125% bile salts) for 10 weeks. The experimental results revealed that fenugreek significantly lowered the incidence of gallstones in mice by 63, 40 and 10% with the incorporation of fenugreek in to HCD at 5, 10 and 15% levels, respectively. The reduction in CGS incidence was mainly due the decline in cholesterol levels of bile as evidenced by the reduction of biliary cholesterol saturation index from 2.57 to 0.77–0.99 with fenugreek incorporation. Besides, fenugreek also showed the regression of pre-established gall stone by 61 and 64% with 6 and 12% fenugreek levels respectively at 10 weeks in mice. The antihypercholesterolemic effect of fenugreek was mainly attributed to the decreased biliary cholesterol saturation index. Our results concluded that the fenugreek seeds not only reduce the induction but also regress the preformed gall stones, thus possess an antilithogenic effect through antihypercholesterolemic action in mice.

Biliary Polyunsaturated Fatty Acids and Telocytes in Gallstone Disease

It has been reported that intake of ω-3 polyunsaturated fatty acids (PUFAs) reduces the risk of coronary heart disease. It also influences bile composition, decreasing biliary cholesterol saturation in the bile of patients with gallstones. In addition to bile composition disturbances, gallbladder hypomotility must be a cofactor in the pathogenesis of cholelithiasis, as it leads to the prolonged nucleation phase. Our current knowledge about gallbladder motility has been enhanced by the study of a population of newly described interstitial (stromal) cells—telocytes (TCs). The purpose of this study was to determine whether TC loss, reported by our team recently, might be related to bile lithogenicity, expressed as cholesterol saturation index or the difference in biliary PUFA profiles in patients who suffer from cholecystolithiasis and those not affected by this disease. We determined biliary lipid composition including the fatty acid composition of the phospholipid species in bile. Thus, we investigated whether differences in biliary fatty acid profiles (ω-3 PUFA and ω-6 PUFA) in gallbladder bile may influence its lithogenicity and the quantity of TCs within the gallbladder wall. We conclude that the altered PUFA concentrations in the gallbladder bile, with elevation of ω-6 PUFA, constitute important factors influencing TC density in the gallbladder wall, being one of the possible pathophysiological components for the gallstone disease development. This study established that altered bile composition in patients with cholelithiasis may influence TC quantity within the gallbladder muscle, and we concluded that reduction in TC number may be a consequence of the supersaturated bile toxicity, while some other bile components (ω-3 PUFA, glycocholic, and taurocholic acids) may exert protective effects on TC and thus possibly influence the mechanisms regulating gallbladder and extrahepatic bile duct motility. Thus, ω-3 PUFA may represent a possible option to prevent formation of cholesterol gallstones.

Prevention of gallbladder hypomotility via FATP2 inhibition protects from lithogenic diet-induced cholelithiasis.

Gallstone disease is a widespread disorder costing billions for annual treatment in the United States. The primary mechanisms underlying gallstone formation are biliary cholesterol supersaturation and gallbladder hypomotility. The relative contribution of these two processes has been difficult to dissect, as experimental lithogenic diets cause both bile supersaturation and alterations in gallbladder motility. Importantly, there is no mechanistic explanation for obesity as a major risk factor for cholelithiasis. We discovered that lithogenic diets induce ectopic triacylglycerol (TAG) accumulation, a major feature of obesity and a known muscle contraction impairing condition. We hypothesized that prevention of TAG accumulation in gallbladder walls may prevent gallbladder contractile dysfunction without impacting biliary cholesterol saturation. We utilized adeno-associated virus-mediated knock down of the long-chain fatty acid transporter 2 (FATP2; Slc27A2), which is highly expressed by gallbladder epithelial cells, to downregulate lithogenic diet-associated TAG accumulation. FATP2-knockdown significantly reduced gallbladder TAG, but did not affect key bile composition parameters. Importantly, measurements with force displacement transducers showed that contractile strength in FATP2-knockdown gallbladders was significantly greater than in control gallbladders following lithogenic diet administration, and the magnitude of this effect was sufficient to prevent the formation of gallstones. FATP2-driven fatty acid uptake and the subsequent TAG accumulation in gallbladder tissue plays a pivotal role in cholelithiasis, and prevention of this process can protect from gallstone formation, even in the context of supersaturated bile cholesterol levels, thus pointing to new treatment approaches and targets.

The NPC1L1 Polymorphism 1679C>G Is Associated with Gallstone Disease in Chinese Patients.

Niemann Pick Type C1 Like 1 (NPC1L1) protein plays a key role in intestinal and hepatic cholesterol metabolism in humans. Genetic variation in NPC1L1 has been widely studied in recent years. We analyzed NPC1L1 single nucleotide polymorphisms in Chinese gallstone disease patients to investigate their association with gallstone disease. NPC1L1 mRNA expression was also measured in liver biopsies from patients with cholesterol gallstone disease and compared between genotypes. The G allele of the g1679C>G (rs2072183) polymorphism was significantly more prevalent in patients with gallstones compared with gallstone-free subjects. Moreover, patients carrying the G allele had lower hepatic NPC1L1 mRNA expression and higher biliary cholesterol (molar percentages) and cholesterol saturation index. Our study suggests that the G allele of the NPC1L1 polymorphism g1679C>G may be a positive marker of gallstone formation risk.

Hypocholesterolemic and choleretic effects of three dimethoxycinnamic acids in relation to 2,4,5-trimethoxycinnamic acid in rats fed with a high-cholesterol/cholate diet

Background2,4,5-Trimethoxycinnamic acid (2,4,5-TMC) is the major and non-toxic metabolite of α-asarone, which retains hypocholesterolemic and choleretic activities. We compared the activities of 2,4,5-TMC with those of 2,4-dimethoxycinnamic acid (2,4-DMC), 3,4-DMC and 3,5-DMC, to understand the role of the methoxyls on carbons 2, 4 and 5 on the pharmacologic properties of these compounds. MethodsThe methoxycinnamic acids were administered to high-cholesterol/cholate-fed rats. We measured bile flow, and quantified bile acids, phospholipids and cholesterol in bile, and cholesterol and cholesterol-lipoproteins in serum. The inhibition of HMG-CoA reductase by the methoxycinnamic acids was evaluated in vitro. ResultsThe four methoxycinnamic acids decreased serum cholesterol, without affecting the concentration of HDL-cholesterol. 2,4,5-TMC produced the highest decrease in LDL-cholesterol, 73.5%, which exceeds the range of statins (20–40%), and produced the highest inhibition of the activity of HMG-CoA reductase. 3,4-DMC produced the highest increase in bile flow, bile acids and phospholipids concentrations, and reduction in bile cholesterol, which led to a decrease in the biliary cholesterol saturation index. Conclusions2,4,5-TMC (which has three methoxyls) had the highest hypocholesterolemic activity, while 3,4-DMC, which lacks the methoxyl in carbon 2 but conserves the two other methoxyls in an adjacent position, had the highest choleretic activity and a probable cholelitholytic activity. In methoxycinnamic acids with two methoxyls in non-adjacent positions (2,4-DMC and 3,5-DMC), the hypocholesterolemic and choleretic activities were not as evident. 2,4,5-TMC and 3,4-DMC, which did not cause liver damage during the treatment period, should be further explored as a hypocholesterolemic and choleretic compounds in humans.

Disruption of the Murine Protein Kinase Cβ Gene Promotes Gallstone Formation and Alters Biliary Lipid and Hepatic Cholesterol Metabolism

The protein kinase C (PKC) family of Ca(2+) and/or lipid-activated serine-threonine protein kinases is implicated in the pathogenesis of obesity and insulin resistance. We recently reported that protein kinase Cβ (PKCβ), a calcium-, diacylglycerol-, and phospholipid-dependent kinase, is critical for maintaining whole body triglyceride homeostasis. We now report that PKCβ deficiency has profound effects on murine hepatic cholesterol metabolism, including hypersensitivity to diet-induced gallstone formation. The incidence of gallstones increased from 9% in control mice to 95% in PKCβ(-/-) mice. Gallstone formation in the mutant mice was accompanied by hyposecretion of bile acids with no alteration in fecal bile acid excretion, increased biliary cholesterol saturation and hydrophobicity indices, as well as hepatic p42/44(MAPK) activation, all of which enhance susceptibility to gallstone formation. Lithogenic diet-fed PKCβ(-/-) mice also displayed decreased expression of hepatic cholesterol-7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8b1). Finally, feeding a modified lithogenic diet supplemented with milk fat, instead of cocoa butter, both increased the severity of and shortened the interval for gallstone formation in PKCβ(-/-) mice and was associated with dramatic increases in cholesterol saturation and hydrophobicity indices. Taken together, the findings reveal a hitherto unrecognized role of PKCβ in fine tuning diet-induced cholesterol and bile acid homeostasis, thus identifying PKCβ as a major physiological regulator of both triglyceride and cholesterol homeostasis.

Changing treatments for bacterial meningitis

The relations between the concentration of deoxycholic acid (DCA), the cholesterol saturation index, and the nucleation time in gall bladder bile were measured to determine the role of DCA in bile in the pathogenesis of cholesterol gall stone disease. Bile was obtained from patients with cholesterol gall stones (n = 30), subjects without gall stones (n = 35), and patients with pigment gall stones (n = 9). Three of 30 cholesterol gall stone patients and 10 of 35 gall stone free subjects were treated with antibiotics by mouth to decrease the concentration of bile DCA and determine the effect of DCA on biliary lithogenecity. Both the percentage and concentration of DCA in bile were similar in patients with and without cholesterol gall stones despite significant differences in their cholesterol saturation indices and nucleation times. Neither the percentage nor the concentration of DCA in bile correlated with either the cholesterol saturation index or the nucleation time. Analysis of subgroups with matching cholesterol saturation indices showed no correlation between the proportion of DCA in the bile and the cholesterol nucleation time. The proportion of DCA in bile was decreased by antibiotic treatment, but this had no effect on the cholesterol saturation index or nucleation time. These results suggest that DCA in bile is not responsible for biliary cholesterol saturation or cholesterol nucleation time.

The Spectrum of Liver Diseases Related toABCB4Gene Mutations: Pathophysiology and Clinical Aspects

Class III multidrug resistance P-glycoproteins, Mdr2 in mice and MDR3 in human, are canalicular phospholipid translocators involved in biliary phospholipid (phosphatidylcholine) excretion. The role of an ABCB4 gene defect in liver disease has been initially proven in a subtype of progressive familial intrahepatic cholestasis called PFIC3, a severe pediatric liver disease that may require liver transplantation. Several ABCB4 mutations have been identified in children with PFIC3 and are associated with low level of phospholipids in bile leading to a high biliary cholesterol saturation index. ABCB4 mutations are associated with loss of canalicular MDR3 protein and /or loss of protein function. There is evidence that a biallelic or monoallelic ABCB4 defect causes or predisposes to several human liver diseases (PFIC3, low phospholipid associated cholelithiasis syndrome, intrahepatic cholestasis of pregnancy, drug-induced liver injury, transient neonatal cholestasis, adult biliary fibrosis, or cirrhosis). Most patients with MDR3 deficiency have a favorable outcome with ursodeoxycholic acid (UDCA) therapy, but some PFIC3 patients who do not respond to UDCA treatment still require liver transplantation. The latter should be good candidates for a targeted pharmacologic approach and/or to cell therapy in the future.

Regression of preestablished cholesterol gallstones by dietary garlic and onion in experimental mice

We have recently reported the health beneficial potential of dietary garlic and onion in reducing the incidence and severity of cholesterol gallstone (CGS) during its experimental induction in mice. In the current study, the efficacy of dietary garlic and onion in regressing preestablished CGS was investigated in experimental mice. After inducing CGS in mice with a lithogenic diet for 10 weeks, they were maintained on basal diets containing 0.6% dehydrated garlic or 2% dehydrated onion for a further 10 weeks. Dietary garlic and onion, either raw or heat processed, regressed preformed CGS in mice up to 53% to 59%, whereas the regression in the basal control diet group was only 10%. The antilithogenic potency of garlic was decreased by its heat processing, but not in the case of onion. Biliary cholesterol was significantly decreased in garlic- and onion-fed animals. Biliary cholesterol saturation index and hydrophobicity index were significantly lowered by dietary garlic and onion. Serum and liver cholesterol levels were decreased by feeding these spices during post-CGS induction period. Hepatic hydroxymethylglutaryl–coenzyme A reductase activity was increased after feeding garlic and onion, whereas activities of the cholesterol-degrading enzymes cholesterol-7 α-hydroxylase and sterol-27-hydroxylase were increased in spice-fed groups. These results indicate that feeding garlic and onion effectively accelerates the regression of preformed CGS by promoting cholesterol desaturation in bile. This observation is significant in the context of evolving dietary intervention strategy to address regression of existing CGS and stopping the possible recurrence.

Effect of dietary garlic and onion on biliary proteins and lipid peroxidation which influence cholesterol nucleation in bile

Formation of cholesterol gallstones in gallbladder is controlled by procrystallizing and anticrystallizing factors present in bile. Dietary garlic and onion have been recently observed to possess anti-lithogenic potential in experimental mice. In this investigation, the role of biliary proteins from rats fed lithogenic diet or garlic/onion-containing diet in the formation of cholesterol gallstones in model bile was studied. Cholesterol nucleation time of the bile from lithogenic diet group was prolonged when mixed with bile from garlic or onion groups. High molecular weight proteins of bile from garlic and onion groups delayed cholesterol crystal growth in model bile. Low molecular weight (LMW) proteins from the bile of lithogenic diet group promoted cholesterol crystal growth in model bile, while LMW protein fraction isolated from the bile of garlic and onion groups delayed the same. Biliary LMW protein fraction was subjected to affinity chromatography using Con-A and the lectin-bound and unbound fractions were studied for their influence on cholesterol nucleation time in model bile. Major portion of biliary LMW proteins in lithogenic diet group was bound to Con-A, and this protein fraction promoted cholesterol nucleation time and increased cholesterol crystal growth rate, whereas Con-A unbound fraction delayed the onset of cholesterol crystallization. Biliary protein from garlic/onion group delayed the crystallization and interfered with pronucleating activity of Con-A bound protein fraction. These data suggest that apart from the beneficial modulation of biliary cholesterol saturation index, these Allium spices also influence cholesterol nucleating and antinucleating protein factors that contribute to their anti-lithogenic potential.

15 Alcohol Intake and Development of Symptomatic Gallstones: An Inverse Association - A UK Prospective Cohort Study

Background. Obesity may predispose to the formation of gallstones by increasing both bile stasis and biliary cholesterol saturation. No prospective studies have investigated this hypothesis in both men and women in a European population. Furthermore, it is unknown if the association may be confounded by higher energy intake or lower physical activity. The current study aimed to investigate this relationship in a UK prospective study and to quantify, for the first time, the effect of Body Mass Index (BMI) adjusted for total energy intake and physical activity. Methods. The EPIC-Norfolk Study (European Prospective Investigation into Cancer) recruited 25, 639 men and women, aged 45-74 years between 1993-1997. Participants completed validated questionnaires assessing physical activity, alcohol consumption and total energy intake. Height and weight were measured at a baseline health examination. The cohort was monitored for participants who developed symptomatic gallstones over a ten-year period. The association between BMI and the risk of developing symptomatic gallstones was estimated using hazard ratios (HR) derived from Cox Regression models, adjusted for sex, age, physical activity, total energy intake and alcohol intake. BMI was modelled both as a categorical (< 25, 25 < 30, 30 < 35 and ≥ 35 kg/m2) and as a continuous exposure. Results. In the cohort, 266 participants (of whom 68.3% were women) developed symptomatic gallstones at a mean age of 61.8 years (range 42.7 76.5) with most presenting with biliary colic. Compared with the normal weight (BMI < 25 kg/m2) group, the hazard ratio for symptomatic gallstones in the 25 < 30 kg/m2 group was 1.61, (95 % CI 1.20 2.17, p=0.002) and was highest in the ≥ 35 kg/m2 group (HR= 3.33 (95 % CI 2.02 5.49, p < 0.001)). Overall, each 1 kg/m2 rise in BMI was associated with an increased hazard of symptomatic gallstones of 8% (95% CI 5 11, p < 0.001). The population attributable risk percent for symptomatic gallstones was 33 % for having a BMI ≥ 25 kg/ m2. Conclusions. Higher BMIwas associatedwith an increased risk of symptomatic gallstones, now reported for the first time in a prospective study in a European population in both men and women. If causal, achieving a BMI of < 25 kg/m2 would be associated with a reduction in incidence of symptomatic gallstones by a third. As the association was still significant after adjusting for physical activity and energy intake, this suggests that adiposity has specific biological mechanisms in the aetiology of gallstones.

14 Body Mass Index and the Risk of Symptomatic Gallstones - A Prospective Cohort Study in a UK Population

Background. Obesity may predispose to the formation of gallstones by increasing both bile stasis and biliary cholesterol saturation. No prospective studies have investigated this hypothesis in both men and women in a European population. Furthermore, it is unknown if the association may be confounded by higher energy intake or lower physical activity. The current study aimed to investigate this relationship in a UK prospective study and to quantify, for the first time, the effect of Body Mass Index (BMI) adjusted for total energy intake and physical activity. Methods. The EPIC-Norfolk Study (European Prospective Investigation into Cancer) recruited 25, 639 men and women, aged 45-74 years between 1993-1997. Participants completed validated questionnaires assessing physical activity, alcohol consumption and total energy intake. Height and weight were measured at a baseline health examination. The cohort was monitored for participants who developed symptomatic gallstones over a ten-year period. The association between BMI and the risk of developing symptomatic gallstones was estimated using hazard ratios (HR) derived from Cox Regression models, adjusted for sex, age, physical activity, total energy intake and alcohol intake. BMI was modelled both as a categorical (< 25, 25 < 30, 30 < 35 and ≥ 35 kg/m2) and as a continuous exposure. Results. In the cohort, 266 participants (of whom 68.3% were women) developed symptomatic gallstones at a mean age of 61.8 years (range 42.7 76.5) with most presenting with biliary colic. Compared with the normal weight (BMI < 25 kg/m2) group, the hazard ratio for symptomatic gallstones in the 25 < 30 kg/m2 group was 1.61, (95 % CI 1.20 2.17, p=0.002) and was highest in the ≥ 35 kg/m2 group (HR= 3.33 (95 % CI 2.02 5.49, p < 0.001)). Overall, each 1 kg/m2 rise in BMI was associated with an increased hazard of symptomatic gallstones of 8% (95% CI 5 11, p < 0.001). The population attributable risk percent for symptomatic gallstones was 33 % for having a BMI ≥ 25 kg/ m2. Conclusions. Higher BMIwas associatedwith an increased risk of symptomatic gallstones, now reported for the first time in a prospective study in a European population in both men and women. If causal, achieving a BMI of < 25 kg/m2 would be associated with a reduction in incidence of symptomatic gallstones by a third. As the association was still significant after adjusting for physical activity and energy intake, this suggests that adiposity has specific biological mechanisms in the aetiology of gallstones.

Dietary garlic and onion reduce the incidence of atherogenic diet-induced cholesterol gallstones in experimental mice

Mice fed with diet containing 0.5 % cholesterol for 10 weeks resulted in cholesterol supersaturation in gallbladder bile which promoted the formation of cholesterol gallstones (CGS). In this study, dietary hypocholesterolaemic spices, garlic and onion (both raw or heat-processed) were examined for their antilithogenic potential by including at 0.6 and 2.0 % level, respectively, along with lithogenic (LG) diet for 10 weeks. Dietary garlic and onion reduced the CGS incidence by 15-39 %, the effect being maximum in the heat-processed onion group. Dietary garlic and onion markedly reduced biliary cholesterol. The cholesterol:phospholipid ratio which was 1.58 in the LG diet group was reduced to 0.73-0.96 in the garlic and onion groups. The biliary cholesterol saturation index was 0.92, 1.25, 1.09 and 0.86, respectively, in the heat-processed onion, raw garlic, heat-processed garlic and raw onion groups, while it was 1.9 in the LG group. The hydrophobicity index of bile was - 0.08, - 0.079, - 0.032 and - 0.073, respectively, in the heat-processed onion, raw garlic, heat-processed garlic and raw onion groups, while it was +0.054 in the LG group. Hepatic hydroxymethyl glutaryl-CoA reductase activity was lowered in the LG diet-fed group, while dietary garlic or onion countered this alteration and also increased the activities of hepatic cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase. Serum and liver cholesterol were decreased by feeding garlic or onion compared to the LG diet. Thus, dietary Allium spices exerted antilithogenic influence by decreasing the cholesterol hyper-secretion into bile and increasing the bile acid output thus decreasing the formation of lithogenic bile in experimental mice.

Inhibiting intestinal NPC1L1 activity prevents diet-induced increase in biliary cholesterol in Golden Syrian hamsters

Niemann-Pick C1-like 1 (NPC1L1) facilitates the uptake of sterols into the enterocyte and is the target of the novel cholesterol absorption inhibitor, ezetimibe. These studies used the Golden Syrian hamster as a model to delineate the changes in the relative mRNA expression of NPC1L1 and other proteins that regulate sterol homeostasis in the enterocyte during and following cessation of ezetimibe treatment and also to address the clinically important question of whether the marked inhibition of cholesterol absorption alters biliary lipid composition. In hamsters fed a low-cholesterol, low-fat basal diet, the abundance of mRNA for NPC1L1 in the small intestine far exceeded that in other regions of the gastrointestinal tract, liver, and gallbladder. In the first study, female hamsters were fed the basal diet containing ezetimibe at doses up to 2.0 mg.day(-1).kg body wt(-1). At this dose, cholesterol absorption fell by 82%, fecal neutral sterol excretion increased by 5.3-fold, and hepatic and intestinal cholesterol synthesis increased more than twofold, but there were no significant changes in either fecal bile acid excretion or biliary lipid composition. The ezetimibe-induced changes in intestinal cholesterol handling were reversed when treatment was withdrawn. In a second study, male hamsters were given a diet enriched in cholesterol and safflower oil without or with ezetimibe. The lipid-rich diet raised the absolute and relative cholesterol levels in bile more than fourfold. This increase was largely prevented by ezetimibe. These data are consistent with the recent finding that ezetimibe treatment significantly reduced biliary cholesterol saturation in patients with gallstones.

Ezetimibe prevents cholesterol gallstone formation in mice

Intestinal cholesterol absorption may influence gallstone formation and its modulation could be a useful therapeutic strategy for gallstone disease (GSD). Ezetimibe (EZET) is a cholesterol-lowering agent that specifically inhibits intestinal cholesterol absorption. To test whether EZET can prevent gallstone formation in mice. Gallstone-susceptible C57BL/6 inbred mice were fed control and lithogenic diets with or without simultaneous EZET administration. Lithogenic diet increased biliary cholesterol content and secretion, and induced sludge or gallstone formation in 100% of the animals. EZET administration reduced intestinal cholesterol absorption by 90% in control animals and by 35% in mice receiving the lithogenic diet. EZET prevented the appearance of cholesterol crystals and gallstones. In addition, mice fed the lithogenic diet plus EZET exhibited a 60% reduction in biliary cholesterol saturation index. Of note, EZET treatment caused a significant increase in bile flow (+50%, P<0.01) as well as bile salt, phospholipid and glutathione secretion rates (+60%, +44% and +100%, respectively, P<0.01), which was associated with a moderately increased expression of hepatic bile salt transporters. In addition, relative expression levels of Nieman-Pick C1 like 1 (NPC1L1) in the enterohepatic axis in humans were assessed. Expression levels of NPC1L1 were 15- to 30-fold higher in the duodenum compared with the liver at transcript and protein levels, respectively, suggesting preferential action of EZET on intestinal cholesterol absorption in humans. In a murine model of GSD, EZET prevented gallstone formation by reducing intestinal cholesterol absorption and increasing bile salt-dependent and -independent bile flow. EZET could be useful in preventing GSD disease in susceptible patients.

Effect of Ezetimibe on the Prevention and Dissolution of Cholesterol Gallstones

Cholesterol cholelithiasis is one of the most prevalent and most costly digestive diseases in developed countries and its incidence has increased markedly in Asian countries owing to the adoption of Western-type dietary habits. Because animal experiments showed that high efficiency of intestinal cholesterol absorption contributes to gallstone formation, we explored whether the potent cholesterol absorption inhibitor ezetimibe could prevent gallstones and promote gallstone dissolution in mice and reduce biliary cholesterol content in human beings. Male gallstone-susceptible C57L mice were fed a lithogenic diet and concomitantly administered with ezetimibe at 0, 0.8, 4, or 8 mg/kg/day for 8 or 12 weeks. Gallbladder biles and gallstones were examined by microscopy. Gallbladder emptying in response to cholecystokinin octapeptide was measured gravimetrically. Biliary lipid outputs were analyzed by physical-chemical methods. Cholesterol absorption efficiency was determined by fecal dual-isotope ratio and mass balance methods. Lipid changes in gallbladder biles of gallstone patients vs overweight subjects without gallstones were examined before (day 0) and at 30 days after ezetimibe treatment (20 mg/day). Ezetimibe prevented gallstones by effectively reducing intestinal cholesterol absorption and biliary cholesterol secretion, and protected gallbladder motility function by desaturating bile in mice. Treatment with ezetimibe promoted the dissolution of gallstones by forming an abundance of unsaturated micelles. Furthermore, ezetimibe significantly reduced biliary cholesterol saturation and retarded cholesterol crystallization in biles of patients with gallstones. Ezetimibe is a novel approach to reduce biliary cholesterol content and a promising strategy for preventing or treating cholesterol gallstones by inhibiting intestinal cholesterol absorption.

Increased expression of LXRα, ABCG5, ABCG8, and SR-BI in the liver from normolipidemic, nonobese Chinese gallstone patients

Cholesterol supersaturation of bile is one prerequisite for gallstone formation. In the present study of Chinese patients with gallstones, we investigated whether this phenomenon was correlated with the hepatic expression of genes participating in the metabolism of cholesterol and bile acids. Twenty-two nonobese, normolipidemic patients (female-male, 11:11) with gallstones were investigated with 13 age- and body mass index-matched gallstone-free controls (female-male, 10:3). The bile from the gallstone patients had higher cholesterol saturation than that from the controls. The mRNA levels of ABCG5, ABCG8, and liver X receptor alpha (LXRalpha) in the gallstone patients were increased by 51, 59, and 102%, respectively, and significantly correlated with the molar percentage of biliary cholesterol and cholesterol saturation index (CSI). The mRNA and protein levels of the hepatic scavenger receptor class B type I (SR-BI) were increased, and a significant correlation was found between the protein levels and the CSI. No differences were recorded between the two groups concerning the hepatic synthesis of cholesterol, bile acids, and esterification of cholesterol. Our results suggest that the upregulation of ABCG5/ABCG8 in gallstone patients, possibly mediated by increased LXRalpha, may contribute to the cholesterol supersaturation of bile. Our data are consistent with the possibility that increased amounts of biliary cholesterol may originate from plasma HDL cholesterol by enhanced transfer via SR-BI.